CASY Cell Counter + Analyser System Model TT Operator … · Cell Counter + Analyser System Model...

CASY®

Cell Counter + Analyser System

Model TT

Operator Manual

Roche Innovatis AG

CASY® is a registered Trademark of Roche Innovatis AG

CASY® Model TT Version 2.3E __________________________________________________________________________________

__________________________________________________________________________________ Roche Innovatis AG • Krämerstr. 22 • D-72764 Reutlingen • ℡ +49 (0) 7121 38786-0 • [email protected]



1. Table of Contents

1. Table of Contents ........................................................................................... 3

2. Introduction .................................................................................................... 5

2.1. The CASY® measuring principle..................................................................................... 5

2.2. CASY® Model TT ........................................................................................................... 6

3. Device configuration ...................................................................................... 6

3.1. Measuring unit.............................................................................................................. 6

3.2. Measuring capillary and external electrode.................................................................... 8

3.3. Pressure system and power supply ................................................................................ 9

3.4. Control panel................................................................................................................ 10

4. Start-up ........................................................................................................... 11

4.1. Choosing the location................................................................................................... 11

4.2. Cabling ......................................................................................................................... 11

4.3. Switch-on ..................................................................................................................... 12

4.4. Preparing for measurement........................................................................................... 13

5. Caring for your CASY® .................................................................................... 14

5.1. Weekly cleaning routine................................................................................................ 15

5.2. Cleaning the cylinders and the sampling chamber ........................................................ 15

5.3. How to set CASY® out of operation.............................................................................. 16

6. Operation of CASY® Model TT ....................................................................... 16

6.1. General......................................................................................................................... 16

6.2. Menu Control - MAIN MENU ........................................................................................ 17

6.3. MAIN MENU - Select Setup ........................................................................................... 18

6.4. MAIN MENU - Edit Setup .............................................................................................. 19

6.5. MAIN MENU - Select Capillary....................................................................................... 25

6.6. MAIN MENU - Set Date and Time.................................................................................. 25

6.7. MAIN MENU - System Settings...................................................................................... 26

6.8. MAIN MENU - Service Menu ......................................................................................... 28



7. Performing and evaluating measurements................................................... 29

7.1. Preparing samples .........................................................................................................29

7.2. Measuring samples and displaying size distributions......................................................30

7.3. Numerical measuring results..........................................................................................31

7.4. Points to be observed when performing measurements ................................................33

7.5. Setting the measuring range and the cursor positions ...................................................38

7.6. Manual cursor control using control panel keys.............................................................40

7.7. Outputting the results ...................................................................................................41

8. Appendix ......................................................................................................... 43

8.1. Error messages ..............................................................................................................43

8.2. Data output via the serial interface................................................................................44

8.3. Technical data on CASY® Model TT. ..............................................................................46

Roche Innovatis AG

Krämerstraße 22

D-72764 Reutlingen

Phone: +49 (0)-7121-38786-0

Fax : +49 (0)-7121-38786-99

Mail: [email protected]

Internet: www.innovatis.com



2. Introduction

CASY®-Technology is a German development based on extensive experience gained in analytical working

methods in the fields of cytobiology, microbiology, biochemistry and physics. Its ergonomic and easy operation

were developed specifically for day-to-day, routine use. Standardized cell counting methods, viability checks,

aggregation correction, volumetric measurement with high measuring range dynamics all make the CASY® an

indispensable tool for the qualitative and quantitative assessment of cell cultures.

2.1. The CASY® measuring principle

CASY®-Technology of Schärfe System combines a proven particle measurement technique, referred to as the

Resistance Measurement Principle, with a modern method of signal evaluation, Pulse area analysis.

Pulse area analysis was developed in 1987 by Schärfe System on the basis of Digital Pulse Processing (DPP), a

process employed for the digital recording of electrical signals for more than 40 years. For cell analysis, DPP is

combined with a high scanning rate and a highly-standardized method of analysing cell signals. This combination

facilitates unprecedented measuring range dynamics and guarantees maximum resolution and excellent

reproducibility for all measuring parameters.

Measurement is performed by suspending the cells in CASY®ton, an electrolyte developed specifically for cell

counting and aspirating them through a precision measuring pore of defined geometry at a constant flow speed.

The precision measuring pore is designed as a hole within an artificial precious stone, which is cast inside the

capillary body. During the measurement process, a pulsed low voltage field with 1 MHz is applied to the measuring

pore via two platinum electrodes. The electrolyte-filled measuring pore represents a defined electrical resistance.

During their passage through the measuring pore, the cells displace a quantity of electrolyte corresponding to their

volume. Since intact cells can generally be considered isolators, an increased level of resistance is achieved over the

measuring pore. This resistance is a dimension for the volume of the cells. By contrast, dead cells whose membrane

no longer acts as an electrical barrier, are recorded by the size of their cell nucleus. It is important that the cells are

passing through the measuring pore individually.

The measuring signal is scanned by CASY® at high frequency. As well as the amplitude of the measuring signal,

CASY® also records the shape of the signal as a whole. CASY® calculates the integral of the measuring signal from

the individual measurements. The calculated signal areas are evaluated by a standardized method and accumulated

in a multi-channel analyser (pulse area analysis). A diameter-linear size distribution with a resolution of 400 display

channels is calculated from a linear-volume, original distribution with 524,288 measuring channels. This

distribution forms the basis for the calculation of all other measuring parameters.



The signal amplifier technology developed specifically for CASY® guarantees excellent measuring range dynamics

(> 1 : 70000 in terms of volume and > 1 : 40 in terms of diameter respectively). Measuring range dynamics is

described as the ratio of the smallest to the largest particles that can be analysed simultaneously. This guarantees

that the entire physical measuring range of the used measuring capillaries from approx. 2% to approx. 80% of the

measuring pore diameter is always in view, from very small debris particles to very large cell aggregates.

2.2. CASY® Model TT

CASY® Model TT combines a high degree of ergonomics with functionality. At the push of a button, the electronics

control the measuring stand, record the measuring data, evaluate the measuring data via a multi-channel analyser,

calculate and display the measuring results on the control panel. All measuring parameters can be set through a

minimum of steps. For each measuring capillary, up to 20 settings can be stored for various cell types or various

applications and remain available the next time the device is switched on. The user can focus hid concentration on

his scientific work.

To output the data, a HP-compatible printer (PCL) can be connected directly to the device. The data for display,

recording and printout purposes can also be exported to MS-Excel™ (PC) through a fully automated process. This

requires CASY®excell, which is available from innovatis AG.

3. Device configuration

CASY® Model TT comprises the following functional units:

• Measuring stand consisting of measuring unit, pressure systems and mains power supply (see Figure 1).

• Measuring capillaries (see Figure 1).

• Control panel with graphic display and 13 control keys (see Figure 2).

Measuring unit, pressure system and mains power supply are accommodated in three separate metal cylinders.

This guarantees maximum protection for the highly-sensitive measuring electronics against interference and

enables the measuring capillaries to be arranged in one large, open sampling chamber.

3.1. Measuring unit

The measuring unit (see Figure 1) contains the measuring electronics, the fluid system and the valves required to

control the measuring process. The close monitoring of the measuring volume (200 µl or 400 µl) required to

determine the concentration is carried out by a light-barrier monitored, calibrated precision vertical tube. CASY®

handles larger measuring volumes (maximum 4 ml) by employing a multiple measurement system. The results of



the individual measurements are accumulated. CASY® operates almost dead-volume free, i.e. the required sample

quantity is only slightly larger than the measuring volume. The control and monitoring of the measuring process

are designed to exhibit maximum protection against interference. Any faults that do occur are automatically

detected by CASY® and notified to the user.

The sampling chamber accommodates the following elements:

• Measuring capillary

• External electrode

• Sample platform

• Waste container (left)

• Storage container (right)

Figure 1: Schematic of CASY® Model TT, measuring stand, frontal view

Base

Waste Container

Calibrated Vertical Tube

Power Supply

Storage Container

External Electrode

Pressure System (concealed)

Light-Emitting Diodes

Sample PlatformPrecision Measuring Capillary

Measuring Unit



The measuring unit is controlled by the control panel (see 3.4 and Figure 2). An inspection window, which also

enables the vertical tube to be monitored visually, is situated above the sampling chamber. The inspection window

accommodates three light-emitting diodes, which indicate the filling level of the vertical tube. The green light-

emitting diode indicates the start of a measurement, the lower red light-emitting diode indicates when the 200 µl

level is reached and the upper red light-emitting diode indicates when the 400 µl level has been reached.

Before the start of each measurement, the contents of the measuring capillary are emptied into the waste

container. The sample volume is then aspirated through the measuring capillary under constant low pressure. The

progress of the meniscus can be tracked in the inspection window. The time required to reach the light barriers

depends on the diameter of the used measuring capillary and is precisely monitored by CASY®. This process

enables potential faults resulting from measuring capillary blockages to be automatically detected. During the

measurement process, the vertical tube is also monitored for disturbances caused by air bubbles.

Blockages can be removed by running a clean cycle, which, in addition to refilling the internal fluid system, also

actively counter-flushes the measuring pore (see 4.4). The required CASY®ton is sucked in from the storage

container and then emptied into the waste container. The entire volume of the internal fluid system is approx. 2

ml.

3.2. Measuring capillary and external electrode

The external electrode (see Figure 1) is from platinum, arranged before the measuring capillary, can rotate freely

and be pulled downwards if necessary.

The measuring capillary (see Figure 1) is from highly resistance plastic. It is secured to the sampling chamber

cover by an cap nut. At the lower end of the capillary body, an artificial precious stone is cast with the precision

measuring pore. The measuring pore has a cylindrical geometry with a diameter to length ratio of 1:1. The axial

arrangement of the hole minimises the risk of blockage and prevents impurities from accumulating inside the

capillary body.

To change the measuring capillary, first carefully rotate the external electrode forwards. Then release the cap nut

and remove it with the measuring capillary by pulling downwards. In order to prevent the measuring capillary

becoming contaminated with grease and dust, try to avoid touching its lower end, which is immersed in the

sample.

To re-assemble, screw in the cap nut manually (do not use a tool)! Ensure that the tube extending inside the

measuring capillary is not buckled or pinched during assembly. Then, carefully rotate the external electrode,

without bending, and return to its original position. Ensure that the background is monitored before commencing

the measurement (see 4.4).



If you have to change the measuring capillaries several times in one day, store the unused ones in particle-free

CASY®ton.

Note:

Unused measuring capillaries must always be cleaned thoroughly before being stored. Dried residues in the

measuring pore can result in subsequent blockages. This can be prevented by placing the measuring capillaries in

CASY®clean and leaving overnight. The following day, flush the measuring capillaries carefully using CASY®clean

and then flush repeatedly with distilled water. Blow the fluid from the measuring capillaries and store in a clean

place.

Caution:

The measuring capillaries should be cleaned using only the CASY®clean agent specified by innovatis AG. Other

agents may destroy the measuring capillaries. Innovatis AG is not liable for any damage caused by using

alternative cleaning agents.

3.3. Pressure system and power supply

The pressure system is accommodated in its own cylinder situated behind the measuring unit (concealed in Figure

1). A no-maintenance vacuum pump, combined with the latest pressure sensors, guarantee that the vacuum

required to aspirate the sample remains constant, irrespective of any external pressure. The pressure system also

interacts with the valves on the measuring unit to control all fluid movements required to empty the vertical tube,

replenish the fluid system and counter-flush the measuring capillary.

The power supply for the CASY® measuring electronics is housed in the cylinder arranged outside to the right (see

Figure 1). Special filters ensure that the mains power supply is afforded maximum interference protection.



3.4. Control panel

The control panel (see Figure 2) consists of a graphic display and a membrane keyboard exhibiting 13 keys. In

measuring mode, the graphic display shows the size distribution and indicates the measuring results. All measuring

and output parameters can be displayed and adjusted through the menu control.

The keyboard is divided into three functional areas. The top right-hand area accommodates the keys used to enter

the measuring and output parameters. The bottom right-hand area accommodates the keys used to control the

measuring unit and output the results to the printer. On the left are the keys used to position the evaluation and

the normalization cursors. All keys have a clearly defined pressure point.

Definition of key functions:

MENU: Starts the menu control.

CANCEL: Cancels a measurement or entry.

ENTER: Confirms an entry.

NEXT: Switches from graphic to numerical display. Continue to next number. Switches to set the second cursor pair.

+ : Increases number / change

selection.

− : Decreases number / change selection.

PRINT: Output to printer and/or serial interface (CASY®excell).

CLEAN: Starts one or several cleaning cycles.

START: Starts a measurement.

L-CURSOR: Positions left cursor

: Shifts cursor to the right.

: Shifts cursor to the left.

R-CURSOR: Positions right cursor

Figure 2: CASY® Model TT, Control Panel

L

CURSORMENU CANCEL ENTER

NEXT

PRINT

CLEANR

CURSOR

START

Graphic-Display



4. Start-up

4.1. Choosing the location

CASY® Model TT is a precision measuring instrument. To ensure fault-free measuring operation, you must choose

the location according to the following criteria:

• Place the instrument on a solid, dry work table that is exposed to neither strong mechanical vibrations nor

high-volume noises (e.g. ultrasonic baths).

• Choose the installation site so that CASY® is protected from mechanical damage and water and solvent

spillage.

• Choose a room that is as smoke and dust-free as possible and that has a constant room temperature of

between 15°C and 32°C

• Do not place CASY® in the vicinity of devices that generate strong electrostatic or electromagnetic fields. This

includes specifically computer monitors that work with high-voltage picture tubes, brush motors, flickering

fluorescent lamps, arcing contacts, water baths, gas chromatographs etc. In the event of any alternative site

being unavailable, maintain the greatest possible distance from such devices.

4.2. Cabling

Connect your CASY® to an interference-free measuring instrument socket. Under no circumstances should you

operate CASY® from the same socket as laboratory equipment that works with high switching loads (autoclaves,

centrifuges, agitators etc.). You must also ensure that all laboratory equipment situated in the same vicinity is

interference-free/fault-free. The following connection cables and/or control elements must be connected.

• Data line for control panel (9-pin sub-D plug).

• Mains power cable.

• Printer, if available (25-pin sub-D plug).

• Serial transfer cable for PC connection (CASY®excell), if available.

All connections are clearly labeled to prevent confusion.

Caution:

Before switching on for the first time, ensure that the mains voltage set on the voltage selector on the rear of the

device is consistent with your mains power supply. If it is necessary to convert the mains power supply, please

observe the information on the primary fuses (see 8.3). Innovatis AG accepts no liability for damage caused by

operating the device from an inappropriate power supply.



4.3. Switch-on

Fitted to the rear panel of CASY® is a green power switch, which when turned to ON, illuminates and indicates that

the device is live. After being switched on, CASY® automatically performs all the required initialisation procedures.

Specifically, the system is subjected to a complete self-test during which the hardware, pressure system and all

memory modules (program, calibration, setups) are checked. The following screen appears when the system is

powered up:

SCHÄRFE SYSTEM

CASY® TT Cell Counter + Analyser System

COMPLETE SYSTEM SELFTEST

Hardware : ---> ok Pressure System: ---> ok Memory : running

When the initialisation procedure is complete, your CASY® Model TT will display an empty size distribution screen.

The previously selected measuring capillary and previously used setup is loaded automatically.

Caution:

Before switching on your CASY®, ensure that the control panel is connected and properly bolted to the measuring

stand. Removing the plug from the control panel when the measuring unit is switched to ON may result in damage

to the graphic display electronics. Innovatis AG accepts no liability for damage caused by severing a live

connection between the measuring unit and the control panel.



4.4. Preparing for measurement

CASY® is factory-calibrated for common cell culture media. Preparing for measurement is therefore limited to the

following steps:

• Replenishing the Storage container with clean CASY®ton.

• Emptying the waste container.

• Fitting (see 3.2) and selecting (see 6.5) the required measuring capillary where necessary.

• Selecting an appropriate setup (see 6.3) or manually inputting the parameters (see 6.4)

• Monitoring the background.

You must monitor the background of your CASY® at regular intervals. Problems resulting from choosing an

unsuitable location and also those relating to impurities in the system manifest themselves in too high a

background.

Background monitoring

1. Place a CASY®cup containing CASY®ton below the measuring capillary.

2. Press and hold the CLEAN key down until the clean counter indicates 3. Releasing the key triggers three clean cycles.

3. Place a new CASY®cup containing CASY®ton below the measuring capillary.

4. Press the START key to initiate a measurement.

Note:

To guarantee that the CASY®cup is fixed, you can rotate the sample platform one turn so that the recess is no

longer pointing forwards. This secures the cup.

Depending on the measuring capillary used, the background should not exceed the following values:

150 µm measuring capillary: < 100 Counts/ml (typically 10 - 50)

60 µm measuring capillary: < 200 Counts/ml (typically 20 -100)

45 µm measuring capillary: See instructions on how to use the 45 µm measuring capillary.

Too high a background may result from impurities in your CASY®ton or not having used a new CASY®cup. Filter

the CASY®ton through a sterile filter with a pore size of <= 0,8 µm and use a new CASY®cup for every

measurement. Repeat steps 1 to 4 with freshly filtered CASY®ton. If the system fails to become clean even after

several attempts, you should carry out the Weekly Cleaning routine (see 5.1). If this exercise fails to produce the

desired result, please check that the location of your CASY® satisfies the criteria indicated in 4.1.



Caution:

You must ensure that all the vessels used to prepare the samples, the CASY®ton used to flush the system and

dilute the samples and also any CASY®cups used are free of grease and particle impurities. For routine

preparation of the measuring samples, use a dispenser (10 ml) with integral filter (<=0,8 µm) wherever possible.

To avoid incorrect measurements, CASY®cups should be used once only.

Instructions on how to use the 45 µm measuring capillary:

The 45 µm measuring capillary has been optimised for microbiological applications. A special process for signal

detection is used to ensure that very small cells near the system’s physically determined lower measuring limit are

reliably recorded. This process works reliably from a minimum concentration of 20,000 cells/ml upwards. The

background displayed when running empty with filtered, de-gassed CASY®ton (up to 3000 counts/ml) is of no

consequence for measurements above the minimum concentration.

Your CASY®ton must be de-gassed on a daily basis for measurements using the 45 µm measuring capillary.

Owing to the higher capillary resistance and the higher measuring vacuum, failure to de-gas may lead to false

measuring results due to gas bubbles being present in the capillary. To de-gas the CASY®ton, you will need an

ultrasonic bath and a vacuum pump (suction cylinder with water jet pump). De-gas for at least 10 minutes to

guarantee fault-free measuring operation.

During the measurement process, you must use the protection shield provided. The protection shield guarantees

that the very small measuring signals recorded by the 45 µm capillary cannot be changed by external interference.

The protection shield is connected to the right or the left ground socket in the roof of the sampling chamber by

the corresponding cable. Before a measurement is started, insert the protection shield into the sampling chamber

up to the limit stop, so that the sample is shielded on all sides.

5. Caring for your CASY®

Your CASY® is designed to be low-maintenance; the fluid system however requires regular attention to guarantee

fault-free work over several years. Remember that you are working with biological material. Residues in the fluid

system or growth of bacteria, algae or yeast cells may lead to blockages in the measuring pore, too high a

background or other faults during operation. To avoid such errors, you must follow the cleaning procedure

described below at least once per week. The recommended agent, CASY®clean, is available from innovatis AG.



Caution:

The system should be cleaned using only the CASY®clean agent specified by innovatis AG. Never use aggressive

cleaning fluids on CASY®. Innovatis AG accepts no liability for damage caused by using other cleaning agents!

5.1. Weekly cleaning routine

1. Fill two CASY®cups with CASY®clean.

2. Place one CASY®cup under the measuring capillary.

3. Replace the storage container holding CASY®ton (right-hand cylinder) with the second CASY®cup.

4. Run three cleaning cycles (hold the CLEAN key down and select 3 cycles).

5. Perform a triple measurement using a measuring volume of 400 µl (START).

6. Leave your CASY® for at least 3 hours in CASY®clean.

7. Replace the cleaning solution with two CASY®cups containing distilled water.

8. Run three cleaning cycles (hold the CLEAN key down and select 3 cycles).

9. Perform a triple measurement using a measuring volume of 400 µl (START).

10. Repeat steps 7 to 9 to remove the cleaning solution completely.

11. Repeat steps 7 to 9 using CASY®ton.

12. Your CASY® is now operational again.

Duty of care:

Never leave samples under the measuring capillary! On completion of a series of measurements, place a CASY®cup

containing fresh CASY®ton under the measuring capillary and run at least 3 clean cycles (hold the CLEAN key

down and select 3 cycles).

Before switching off, always monitor the device’s background (see 4.4). Run a measurement using fresh CASY®ton.

5.2. Cleaning the cylinders and the sampling chamber

Clean the surface of your CASY® regularly using a damp micro-fibre cloth. It is important to remove salt solution

spillages immediately. To do this, raise the sample platform and remove by pulling forwards. Methylated spirit can

be used to remove organic impurities. Under no circumstances, however, may other organic solvents be used.



5.3. How to set CASY® out of operation

If your CASY® has not to be operated for several weeks, the fluid system should be drained. Before shutting down,

the fluid system must be cleaned (see 5.1, Weekly Cleaning Routine). Follow this procedure to shut-down the

system:

1. Carry out steps 1 to 10 of the Weekly cleaning routine.

2. Replace the two CASY®cups containing distilled water with two empty CASY®cups.

3. Select the Service Menu option from the main menu.

4. Execute the Dry Liquid System command from the Service Menu.

5. Empty the waste container and the two CASY®cups.

6. You can now switch off your system.

7. Ensure that the system is adequately protected from dust whilst it is shut down.

8. You should begin the Start-up procedure with the Weekly cleaning routine.

Caution:

You must ensure that when the CASY® fluid system contains a saline solution, it is not allowed to dry out. This will

prevent malfunction or damage to the fluid system and valves caused by salt crystals.

6. Operation of CASY® Model TT

6.1. General

CASY® is operated using 13 control keys. Operation is divided into three distinct areas, each of which reflects the

spatial arrangement of the keys (see Figure 2.).

• Entering parameters

Upper right-hand keys: MENU, CANCEL, ENTER, NEXT, +, -

• Measurement operation and output of results

Lower right-hand keys: START, PRINT, CLEAN.

• Evaluation of the size distribution with various cursor positions

Left-hand keys: L-CURSOR, , , R-CURSOR.

Because up to 20 device settings can be permanently stored for each measuring capillary, parameter setting in

most cases can be done by selecting an appropriate setup. Each time CASY® is switched on, the previously used

setup and the previously used measuring capillary are loaded. All other setups are readily available through the



menu function. In addition, the measuring and output parameters can be modified to match the actual

measurement and do not have to be stored in one of the setups.

6.2. Menu Control - MAIN MENU

All CASY® settings are selected with the MENU key. Pressing MENU displays the MAIN MENU.

MAIN MENU TT-2AB-9999 15.04.2002 10:15 ---------------------------------------- * Select Setup

Edit Setup

Select Capillary

Set Date and Time

System Settings

Service Menu

---------------------------------------- USE MENU TO SELECT AND PRESS ENTER TO OPEN. USE CANCEL TO LEAVE SCREEN.

All menu control screens have the same structure. The name of the screen in upper case letters is contained in the

left part of the header, which is separated by a horizontal line: MAIN MENU. Displayed to its right are the serial

number of your CASY®, the date and time. The lower border, also separated by a horizontal line, contains

instructions on how the screen and individual keys operate.

Use the MENU key to select individual menu items. Pressing ENTER opens a menu item. Pressing CANCEL closes

the MAIN MENU. If you overshoot the item you wish to select, you can move backwards by pressing and holding

down the MENU key.

Irrespective of which menu control screen you are in, pressing CANCEL repeatedly returns you to the MAIN

MENU and then to the size distribution display.



6.3. MAIN MENU - Select Setup

The Select Setup option opens the SETUP SELECTION screen. This screen enables you to select one of 20

permanently stored setups for measuring capillary currently in use.

SETUP SELECTION TT-2AB-9999 15.04.2002 10:15 ----------------------------------------

Capillary: 150 µm

Setup No: 00 01 02 03 04 05 06 07 08 09

*

10 11 12 13 14 15 16 17 18 19

---------------------------------------- USE NEXT TO SELECT SETUP AND PRESS ENTER TO LOAD. USE CANCEL TO LEAVE SCREEN.

The setups are numbered from 00 to 19. Each setup stores a complete set of parameters for the selected

measuring capillary. The setups already in use to store parameters are tagged with . If no setup is logged, the

system works with the factory-default set of parameters.

Pressing the NEXT key selects a setup. You may select only those setups that have been logged and tagged with

. Pressing and holding down the NEXT key enables you to move the selector mark backwards. Pressing ENTER

displays the selected setup.

Further details on the setup screen can be found under Edit Setup (see 6.4). Check whether the selected setup is

matching your needs. Beside the measuring and evaluation parameters, each setup has a distinct name which you

can define and which is displayed in the top right-hand corner of the setup screen.

Press ENTER again to load the setup displayed. If you do not wish to load the setup displayed, you can return to

Setup Selection by pressing CANCEL.

Note:

The setup parameters cannot be changed at this point. To edit a loaded setup, use the Edit Setup menu item

described below (see 6.4).



6.4. MAIN MENU - Edit Setup

Edit Setup opens the setup screen. All the parameters provided by CASY® to define the measuring conditions,

display the size distribution, calculate and output the measuring results, can be edited in this screen. The active

parameters are always displayed. Any setup you wish to edit must first be loaded through Select Setup (see 6.3).

SETUP NUMBER: 01 NAME: Chondrozyt ---------------------------------------- Capillary : 150 µm X-Axis : 50 µm Sample Vol : 400 µl Cycles : 2 ---------------------------------------- Dilution : 1.000e+02 Y-Axis : Auto Eval.Cursor : 11.20 - 50.00 µm Norm.Cursor : 6.80 - 50.00 µm %Calculation: %Via Debris : On Aggr.Correct: Auto P.Vol: 0.000e+00 fl Interface : Par P.Feed : On Print-Mode : Manual Graphic: On ---------------------------------------- USE MENU AND NEXT TO STEP. USE +/- TO EDIT. USE CANCEL TO LEAVE SCREEN OR ENTER TO CONFIRM CHANGES.

The setup screen is divided into three areas. The header, separated by a horizontal line, contains the SETUP

NUMBER (00-19) and the NAME (setup name) you have defined. If the active parameters do not correspond to

any of the stored setups, two forward slashes // are shown in place of the setup name and NOT SAVED in place

of the setup name.

The block below, separated by another horizontal line, contains all the measuring parameters. These parameters

must be defined before a measurement takes place. They may not be retrospectively changed.

The main block below contains all evaluation parameters and also the output parameters, which define the

settings for outputting via a directly connected printer or via the serial interface. The parameters can be modified

following a measurement.

When a setup screen is opened, the input marker is located initially on the Dilution input field. The current input

position is indicated by a flashing, horizontal line below the characters which can currently be changed. From this

point, you can use the MENU key to move forwards or backwards between the input fields. To move forwards,

press the MENU key briefly. The input marker skips forward to the next available input position. To move



backwards, keep the MENU key pressed until the input marker reaches the position you require. Both methods

move you to the start or the end of the screen when the last or the first parameter respectively is reached.

When inputting numbers, you can move forwards from number to number using the NEXT key. To increase or

decrease a number selected using NEXT, use the + and – keys.

For all other parameters, the + and - keys can be used to select various pre-defined values. This also applies for the

individual characters of the setup name.

NAME (Setup name)

Each setup can be assigned a specified name. Each name can be up to 10 characters long (letters and numbers).

The setup name is output with the numerical measuring results both on the display and on the printout.

Measuring parameters

The measuring parameters must be set before the measurement takes place. They cannot be modified once the

measurement has been carried out. To change the measuring parameters, the sample must always be subjected to

a second measurement!

Capillary: The capillary belonging to the processed setup is displayed here for information only. You cannot

change its value. To select a different capillary, you must use the Select Capillary command in

the Main Menu.

X-axis: The range of the size distribution to be shown in the graphic display can be set in increments of 5

µm or 10 µm. An additional range of 0 - 3 µm is available for the 45 µm measuring capillary. All

scales start at 0 µm. This guarantees optimum comparability of the measuring results.

Sample Vol: Enter here the measuring volume (200 µl or 400 µl). The set measuring volume is automatically

considered as the cell concentration is calculated. For all measuring capillaries with a pore size >=

100, you should in principle use the 400 µl setting.

Cycles: Enter here the number of repeat measurements (1-10). The individual measurements are

automatically accumulated. The maximum measuring volume is 4.0 ml (10 x 400 µl). The set

measuring cycles are automatically considered as the cell concentration is calculated.



Evaluation parameters

Dilution: The dilution factor is used to calculate the cell concentration in the original sample. It is entered in

exponential format (e.g. for dilution 200, enter: 2.000e+02). Pressing NEXT enables the input

marker to move from number to number. Holding this key down causes the input marker to move

backwards.

Y-axis: The Auto setting automatically modifies the Y-axis scale according to the distribution maximum.

The Y-axis scale can also be set in predefined steps between 10 and 7500. This enables the scale

to be manually modified to the level of the distribution maximum. It is advisable to modify

manually, if the peak of interest within the size distribution is not the maximum.

Eval.Cursor: The right and left-hand evaluation cursors (shown in the size distribution as a continuous line)

determine the size range to be used to calculate the measuring result. For all percentage

calculations (% viability, % counts or % volume), the evaluation cursors define the size range for

which the percentage of the standard range (see Norm.Cursor) is calculated. The + and – keys

can be used to position the evaluation cursor. Pressing NEXT enables you to shift the input marker

between the numbers before and after the decimal points. If the marker is positioned before the

decimal point, + and – causes a change by 10 measuring channels, if it is positioned after the

decimal point, the keys cause a change by one measuring channel. One measuring channel

corresponds to 1/400 of the scale range selected under the X-axis (see measuring parameter). In

order to modify the evaluation cursor whilst visually monitoring the size distribution, you may also

use the control panel arrow keys (see 7.6).

Norm.Cursor: The right and left-hand normalization cursors (shown in the size distribution as a dotted line)

determine the size range for which all percentage calculations indicate 100% (% viability, %

counts or % volume). Parameters Total/ml, TotCnt/ml and TotVol/ml are also calculated for this

range. In the % viability setting, the left-hand normalization cursor also defines the size range for

determining Debris/ml. The procedure for setting the normalization cursor is the same as that

described for the evaluation cursor. The normalization cursor can also be modified using the

control panel arrow keys whilst visually monitoring the size distribution.



%Calculation: In addition to the Off setting (percentage calculation disabled), three other percentage calculation

settings (%Via, %Cnt and %Vol) are available.

%Via: This setting calculates the viability of a cell sample in percentage terms. It is assumed that

the evaluation cursor is set to the viable cell size range and the normalization cursor covers the

size range of all cells (dead + viable).

%Cnt: This setting calculates the percentage counts in the size range of the evaluation cursor in

relation to the size range of the normalization cursor, which is set to 100%.

%Vol: This setting calculates the percentage bio volumes in the size range of the evaluation

cursor in relation to the size range of the normalization cursor, which is set to 100%.

Debris: This parameter can be either On or Off. It can be set to On only if parameter %Via is selected

from %Calculation. When set to On, all counts left of the normalization cursor are summed up

and output as debris/ml taking into account measuring volume and dilution factor.

Aggr.Correct: This parameter defines whether and how the aggregation correction of your count results

should be performed. Off, Manual and Auto are the available settings. In the Auto setting, the

aggregation factor of a sample is automatically calculated as the quotient of mean volume and

peak volume. The calculation is based on the following assumptions:

a) The evaluation cursors are set to the viable cell size range, including all aggregates.

b) The size distribution of the viable cells exhibits a clear maximum.

c) The measurement is supported by adequate statistical reliability (> 1000 count results in the

viable cell range).

If your samples do not match Item b) and/or c), you can specify the volume of the individual cells

manually. To do this, you must select the manual setting and enter the mean individual volume of

your sample using parameter P.Vol (see below). This setting is always recommended if the volume

of your cells does not change significantly during the course of an experiment. The aggregate

correction facility is normally used in conjunction with the %Via percentage calculation.

P.Vol: This parameter defines the mean volume of individual cells in your samples for the manual

aggregation correction (see Aggr.Correct) in terms of femtolitres. The value is entered in

exponential format.



Output parameters

Interface: You can specify through the interface, whether after pressing PRINT, the data should be output

through the parallel output – Par, through the serial output – Ser or through both simultaneously

– P + S. The P + S setting enables you print the data, whilst simultaneously transferring it to a

connected computer. The connected printer must be PCL capable. Hewlett-Packard printers and

all compatible products satisfy this requirement. To export data to a PC, innovatis AG offers

CASY®excell, a program which exports and displays your measuring results directly to MS-Excel™.

More precise details on the serial transfer of data can be found in the Appendix (see 8.2).

P.Feed: This parameter can be either On and Off. When set to On, a page is output to the printer after

each print command. When set to Off, the first page is sent to print only when full. For outputting

numerical results (Graphic to Off) and printing setups, this setting allows up to three printouts to

fit onto one page. You can print the page at any time in advance with the Page Feed command

on your printer. When the size distribution is printed (Graphic to On), P.Feed is ignored.

Print Mode: The print mode can be set to either Manual or Auto. When set to Auto, the data is automatically

output after every measurement. When set to Manual, you must press PRINT after a

measurement to output the data.

Graphic: This setting determines whether the size distribution is also to be printed as data is output to the

printer. The only possible settings are On and Off.

You can abort parameter input at any time by pressing either ENTER or CANCEL. If you press CANCEL, your

entries are discarded and the previous settings are restored. Pressing PRINT will printout all setup parameters for

reference purposes. Pressing ENTER gives you the option of saving your new settings in one of the 20 setups.

Note:

A warning message will appear if a measurement is shown in the display and you have changed one of the

measuring parameters X-axis, Sample Vol or Cycles. You will be reminded that the current measuring result may

be lost if you confirm the message with ENTER. At this point, you can press CANCEL to return to the setup screen.

The changes you have made to the measuring parameters will then be reset.



SAVING SETUP TT-2AB-9999 15.04.2002 10:15 ----------------------------------------

Capillary: 150 µm

Setup No: 00 01 02 03 04 05 06 07 08 09

*

10 11 12 13 14 15 16 17 18 19

---------------------------------------- USE NEXT TO SELECT SETUP AND PRESS ENTER TO SAVE. USE CANCEL TO WORK WITH NEW PARAMETERS WITHOUT SAVING.

You must save your entries if you want the settings to be available next time you switch on CASY®. To do this,

press NEXT to select one of the 20 setups and then press ENTER. The setup currently loaded will be offered as a

default setting. You may select both setups that have already been used and tagged with and also unused

setups. If you select a setup tagged with , its stored settings will be overwritten with your new settings. If you

select an unused setup, it will appear tagged with and will be available for selection next time Select Setup is

opened.

If you do not wish to save your settings, press CANCEL. The new settings are used until you select an alternative

setup or switch off your CASY®. They will then no longer be available. As a setup name for unsaved settings, NOT

SAVED appears and the setup number is replaced by two forward slashes //.

Note:

If you try to overwrite a logged setup, a warning message will appear. You will be reminded that pressing ENTER

will permanently delete this setup’s existing settings. Pressing CANCEL enables you to return to the Saving Setup

screen in order to select a free setup for saving.



6.5. MAIN MENU - Select Capillary

The Select Capillary option opens the CAP SELECTION screen that enables you to switch the system to an

alternative capillary size. Depending on how your system is configured, between one and three entries are available

for different measuring capillaries.

CAP. SELECTION TT-2AB-9999 15.04.2002 10:15 ----------------------------------------

Capillary: 150 µm 60 µm

*

---------------------------------------- USE NEXT TO SELECT CAPILLARY AND PRESS ENTER TO LOAD. USE CANCEL TO LEAVE SCREEN.

Pressing NEXT enables you to switch between the measuring capillary sizes available on your system. The selected

measuring capillaries are tagged with . Press ENTER to load the calibration for the selected measuring capillaries.

The setup previously used with this measuring capillary is activated. Please note that previously displayed

measuring data are lost when a capillary setting is changed. Pressing CANCEL enables you to exit the screen

without changing the settings.

6.6. MAIN MENU - Set Date and Time

Your CASY® has an integral Real-Time-Clock. The Set Date and Time function opens the DATE AND TIME

screen, in which you can set date and time. You have the option of changing the date display from EU format

“DD.MM.YYYY” to US format “MM/DD/YYYY”.

Use the MENU key to move between settings and press NEXT to skip to the individual figures used in the date and

time. Use the + and – keys to change the settings. Press ENTER to confirm your settings or CANCEL to exit the

screen without accepting any changes.



DATE AND TIME TT-2AB-9999 15.04.2002 10:15 ----------------------------------------

Date Format: dd.mm.yyyy

Date: 15.04.2002

Time: 10:15:00

---------------------------------------- USE MENU AND NEXT TO STEP. USE +/- TO EDIT. USE CANCEL TO LEAVE SCREEN OR ENTER TO CONFIRM CHANGES.

6.7. MAIN MENU - System Settings

System Settings contains settings relevant to the entire system. The Graph Parameter function allows you to

specify up to four measuring parameters that you wish to be displayed in the upper section of the size distribution

screen. The Used Setups for Capillary function allows you to delete setups not needed anymore.

SYSTEM SETTINGS TT-2AB-9999 15.04.2002 10:15 ---------------------------------------- 1. Graph Parameter: Conc. Range CON 2. Graph Parameter: Aggregation AGG 3. Graph Parameter: Viab.Cells/ml LML 4. Graph Parameter: %Viability VIA Used Setups for Capillary 150 µm: Setup No: 00 01 02 03 04 05 06 07 08 09 * * * * 10 11 12 13 14 15 16 17 18 19 * * ---------------------------------------- USE MENU AND NEXT TO STEP. USE +/- TO EDIT. USE CANCEL TO LEAVE SCREEN OR ENTER TO CONFIRM CHANGES.



All measuring results, date and time and the used setup can be selected as Graph Parameters. Each parameter

has its own abbreviation consisting of up to three characters, which is used to display the relevant value in the size

distribution screen. The following values are available:

Bezeichner Abkürzung Bezeichner Abkürzung Bezeichner Abkürzung

not used --- Viab.Cells/ml LML Peak Diameter PDI

Conc. Range CON %Viability VIA Mean Volume MVL

Counts CNT Debris/ml DML Peak Volume PVL

Counts > C> Volume/ml VML Date DAT

Aggregation AGG %Volume %V Time TIM

Counts/ml CML Total/ml TML Setup SET

%Counts %C Mean Diameter MDI

You can step to the settings of the 4 parameters by pressing MENU. The required parameters are selected from

the predefined values as indicated in the table using the + and – keys.

If not used is selected, the corresponding position in the upper section of the size distribution screen remains

empty.

To delete redundant setups, use the MENU key to set the input marker in the area called Used Setups for

Capillary…. You can only delete setups for the measuring capillary currently selected using Select Capillary. The

diameter of the active measuring capillary is displayed behind Capillary. Pressing NEXT enables you to shift the

input marker between all setups tagged with . Hold the NEXT key down to move the selection marker

backwards. If you wish to delete a setup, press the - key. The setup to be deleted is now tagged with D instead of

. You can cancel your selection at any time by pressing the + key at the relevant position. The D is replaced by .

Follow this procedure to select all the setups you wish to delete.

When you have made all the changes you require, press ENTER to accept the new settings. Pressing CANCEL

enables you to leave the screen and return to the old settings.

Note:

If you have selected certain setups for deletion, a warning message will appear. You will be reminded that pressing

ENTER will permanently delete these setups tagged for deletion. Pressing CANCEL enables you to return to the

SYSTEM SETTINGS screen to revise your selection. Deleting the currently active setup will also delete the current

measuring data.



6.8. MAIN MENU - Service Menu

The Service Menu contains the Dry Liquid System command you require to shut-down your CASY® properly (see

5.3). It also contains two menu items that enable you to check that the system electronics are functioning correctly.

SERVICE MENU TT-2AB-9999 15.04.2002 10:15 ----------------------------------------

* Dry Liquid System Start Selftest Generate Testpattern

---------------------------------------- USE MENU TO SELECT AND PRESS ENTER TO START. USE CANCEL TO LEAVE SCREEN.

Start Selftest performs the self-test known from System Start (see 4.3). This serves to monitor the hardware, the

pressure system and all memory functions in the system (program, calibration, setups). The individual checks are

confirmed by displaying OK. If an error is detected, the corresponding error message will appear (see 8.1).

The Generate Testpattern feature creates a test size distribution and performs a standardized evaluation using

specified parameters. You will find a test printout, generated during the final factory check, in your system

documentation. This command verifies that the evaluation electronics function properly and all calculations

produced by the evaluation software are correct.

Use the MENU and ENTER keys to select and start the individual commands. Pressing CANCEL enables you to exit

the Service Menu.



7. Performing and evaluating measurements

Working with your CASY® is very simple. The system is ready to start a measurement as soon as it is switched

on. However, please observe the following before you start to measure your samples:

• On initial start-up or after moving the system, check the points cited in Section 4.1 (Choosing the location).

And Section 4.2 (Cabling).

• Follow the steps described in Section 4.4 (Preparing for measurement). Specifically:

- Fitting (see 3.2) and selecting (see 6.5) the required measuring capillary where necessary.

- Selecting an appropriate setup (see 6.3) or manually inputting the parameters (see 6.4).

- Background monitoring (see 4.4).

Please note that after power-up, the system will automatically load the previously used setup for the previously

used measuring capillary. Further information on how to manually specify parameters or create a new setup can be

found in Section 7.5.

7.1. Preparing samples

• Fill a CASY®cup with 10 ml CASY®ton.

• Add an aliquot of the cell suspension to be examined (normally between 10 and 200 µl).

• Secure the lid and mix the sample by tilting the CASY®cup three times. Carefully avoid the formation of

bubbles or foam!

Notes:

a) Your CASY® can measure only the samples you provide. The quality of the measuring result depends on how

well you prepare your samples. Standardize the sample preparation procedure as far as possible (used cell

culture media, timing of sample preparation, duration and intensity of cell re-suspension, carrying out of the

required dilution steps etc.)

b) You should always freshly prepare your samples just before the measurement. Depending on the sensitivity

of the cell type, there is no guarantee that your cells in CASY®ton will remain unchanged in terms of cell

count, cell volume, aggregarion and viability over long periods. If a sample remains in CASY®ton for longer

than two minutes, you must remix it before the measurement.

c) Always use new CASY®cups. This ensures that your measuring results cannot be impaired by high

background caused by contaminated measuring cups.



7.2. Measuring samples and displaying size distributions

• Rotate the recess in the sample platform until it points forwards and remove the CASY®cup from below the

measuring capillary.

• Place the sample to be measured below the measuring capillary and ensure that the external platinum

electrode is immersed in the sample.

• Rotate the sample platform somewhat to the side again.

• Press START to perform a measurement using the current parameters. CASY® automatically works through

all the necessary steps. After a short term, the size distribution for your sample appears in the display.

This example shows the size distribution for a eucaryotic cell line.

• To the left of the left-hand normalization cursor appearing as a dotted line (< 7.6 µm), you will see the

Debris Peak.

• Between the left-hand normalization cursor and the left-hand evaluation cursor shown by a solid line (7.6 µm

to 12.8 µm), you will see the peak for the dead cells. In the case of dead cells, the cell membrane no longer

acts as an electrical barrier. Therefore dead cells are shown by the size of their nucleus.

• To the right of the left-hand evaluation cursor, the peak for the viable cells begins followed by cell

aggregates.

Note:

The size distribution for your samples may appear differently, depending on your applications, your cells and the

used measuring capillaries.



7.3. Numerical measuring results

Depending on the Graph Parameters set on your system (see 6.7), you will find the most important measuring

results at the top of the size distribution. You will also see, in the top right-hand corner, the current setting of the

evaluation cursors CL and CR and the normalization cursors NL and NR, which are used as a basis to calculate the

measuring results.

Pressing NEXT enables you to display the entire numerical evaluation of the measurement data. Pressing NEXT

again will return you to the size distribution.

SET : TUMOR-03¦DIL : 5.000E+01 ¦CAP : 150 µm DATE: 15.04.2002¦SVOL: 3 x 400 µl¦SCAL: 40 µm TIME: 10:15¦AGG : MANUAL ¦NORM: 7.60- 40.00 µm SN : TT-2-XX-9999¦PVOL: 4.249E+03 fl¦EVAL: 12.80- 40.00 µm ------------------------------------------ Counts : 2911 ¦ConcRng: OK ¦ >###µm/ml: 1.500e+02 ¦Aggreg : 1.157 ¦ Viable/ml: 1.684e+05 ¦%Viable: 83.04 % ¦ Total/ml : 2.028e+05 ¦ ¦ Debris/ml: 7.483e+04 ¦ ¦ Volume/ml: 1.431e+07 fl¦ ¦ MeanVol : 4.917e+03 fl¦MeanDia: 21.10 µm ¦ PeakVol : 4.250e+03 fl¦PeakDia: 20.10 µm

The numerical results display is divided into two distinct areas. The header, which is separated by a horizontal line,

contains a complete documentation on all measuring parameters used for the measurement. The main section

contains all the measuring results for the set cursor ranges.

Note:

This example shows the format of the numerical display for settings %Calculation = %Via and Aggr.Correct =

Manual. Details on possible settings can be found in Section 6.4. Depending on your settings, some of the

parameters visible in this example may not appear and/or alternative parameters are shown!



Measuring parameters in the header of the numerical display

SET: The setup used for the measurement in NAME-## format. If the current setup is not stored, NOT SAVED will appear here.

TIME: The time at which the measurement was performed.

DATE: The date on which the measurement was performed.

SN: The serial number of your CASY®.

DIL: The dilution factor used to calculate the sample concentration.

SVOL: The sample volume used for the measurement, indicated as cycles x measuring volume.

AGG: The type of aggregation correction used (Off, Manual or Auto).

PVOL: The peak volume entered for the manual aggregation correction. This value is not shown if

the aggregation correction is set to Off or Auto.

CAP: The diameter of the used measuring capillary.

SCAL: The scale of the X-axis (size axis) in µm.

NCUR: The position of the two normalization cursors used for data evaluation.

ECUR: The position of the two evaluation cursors used for data evaluation.

Measuring results for the active cursor settings

The calculation of the aggregation factor, the viable cell concentration, the percentage viability, the total cell

concentration and the debris concentration assumes that the evaluation cursor is set to the size range of the viable

cells, including all aggregates and that the normalization cursor is set to the size range of all cells (dead + viable)

(see 6.4 and 7.5). The actual measured sample volume and the entered dilution factor are taken into account in

the calculation of all values indicated in ml.

Counts: Number of particles/cells counted in the size range of the evaluation cursors.

ConcRng: Indicates whether the measured concentration lies within the permissible range. Possible

indications are Ok or Too High. The permissible maximum concentration depends on the

used measuring capillaries (see below).

> ###µm/ml: ### indicates the current scale of the X-axis (size axis). The parameter indicates the particle

concentration in the size range above the size distribution shown. It can be used to check

that the correct size scale has been selected.

Aggreg: The aggregation factor calculated for the sample from the mean volume and peak volume.

Depending on the Aggr.Correct parameter setting, the aggregation correction is calculated

from the peak volume currently determined or the manually entered peak volume.

Viable/ml: Concentration of viable cells in the sample in the evaluation cursor size range.

%Viable: Percentage of viable cells in the sample.



Total/ml: Total cell count of the sample (dead + viable) in the normalization cursor size range.

Debris/ml: Concentration of particles in the size range to the left of the left-hand normalization cursor.

Volume/ml: Concentration of particle/bio-volume in the evaluation cursor size range.

MeanVol: Mean volume of all particles/cells in the evaluation cursor size range.

MeanDia: Mean diameter of all particles/cells in the evaluation cursor size range.

PeakVol: Volume at the maximum of the size distribution expressed in fl in the evaluation cursor size range.

PeakDia: Maximum of the size distribution expressed in µm in the evaluation cursor size range.

Depending on the selected setting (see 6.4), the following parameters may also be displayed:

Counts/ml: Particle concentration in the evaluation cursor size range.

%Counts: Percentage of particles in the evaluation cursor size range relative to the normalization cursor size range (100%).

TotCnt/ml: Particle concentration in the normalization cursor size range.

%Volume: Percentage of volume in the evaluation cursor size range relative to the normalization cursor size range (100%).

TotVol/ml: Volume in the normalization cursor size range.

Note:

The calculation of the peak diameter PeakDia and the peak volume PeakVol is based on a sliding weighted mean

value. The algorithm will perfectly match each peak width through a five-stage iteration and guarantees an

accurate calculation of the maximum, even for very flat peaks and for measurements with low cell concentrations.

The peak volume determined in this way is also used to automatically calculate the aggregation factor.

7.4. Points to be observed when performing measurements

For the measuring operation, use only CASY®ton, CASY®cups and CASY®clean specified for the CASY® by

innovatis AG. Using alternative measuring solutions, cups and cleaning agents may lead to incorrect measuring

results and cause damage to the CASY® fluid system. Innovatis AG accepts no liability for damage caused by

using alternative consumables.

A smooth measuring operation depends on the system undergoing the regular Weekly Cleaning Routine (see

5.1.) using CASY®clean (see Section 5.1.)!

You must ensure that your CASY® is also kept clean between cleaning cycles. Specifically that cells never remain in

the fluid system for long periods! The daily background monitoring procedure (see 4.4) and the visual check of the

vertical tube will indicate when the system is becoming contaminated.



The vertical tube is a calibrated glass pipette through which CASY®, controlled by a light barrier, aspirates the set

measuring volume. The vertical tube is visible in the inspection window at the front of the measuring unit. During

the measurement, you can visually track the rising level of the fluid meniscus. Depending on the level in the vertical

tube, one or several light diodes (Green = start of measurement, Red 1 = 200 µl measuring volume, Red 2 = 400

µl measuring volume) illuminates. Judge the status of the vertical tube as you would a glass pipette. If you see that

the fluid is no longer running cleanly and drops of fluid remain suspended on the edge, the calculation of your

sample concentration may be adversely affected. In such cases, run a cleaning cycle using CASY®clean (see 5.1.).

Changing the samples

Provided you measure samples of the same cell type in a similar concentration range, you do not have to flush the

system when the samples are changed. If you expect larger concentration differences or wish to perform

measurements with a different sample type, before a new measurement, place a CASY®cup containing freshly

filtered CASY®ton below the measuring capillary and flush the system using the CLEAN function .

Sample concentration

For correct measurement results the concentration of the diluted cell suspension in the CASY®cup should not

exceed certain maximum values. The concentration limit depends on the size of the measuring capillary:

150 µm measuring capillary: < 20 000 counts/ml

60 µm measuring capillary: < 100,000 counts/ml

45 µm measuring capillary: < 250,000 counts/ml

Higher concentrations than specified result in coincidence errors, i.e. an increased probability that two particles

will be in the measuring pore at the same time. CASY® will evaluate two particles being in the measuring pore at

the same time as if they were one particle with a correspondingly larger volume. The calculated particle count will

therefore be too low and the size distribution will shift towards the larger volumes.



Note:

The coincidence limits do not depend on to the positions of your evaluation cursor settings. They are always

determined for the entire physical measuring range of the used measuring capillary (approx. 2% to 80% of the

measuring pore diameter). In the case of cell samples, this includes cell debris as well as cell aggregate with a

diameter above the scaling range selected for the size axis (X-axis). This guarantees that the cell count and cell

volume are always measured correctly.

You are not required to check yourself whether the concentration of your samples is too high. CASY® displays the

following warning message if the coincidence limit values indicated above are exceeded.

ERROR SCREEN TT-2AB-9999 15.04.2002 10:15 ----------------------------------------

Concentration too high !

Please dilute your sample Measured Counts/ml : 34345 Max Counts/ml allowed : 20000

Further information will be Provided by your manual!

---------------------------------------- USE ENTER TO CONFIRM. PRESS CLEAN TO FLUSH THE LIQUID SYSTEM.

You must confirm this message by pressing ENTER. The measuring result is displayed, but the ConcRng parameter

is shown as Too high. To obtain valid measuring results (cell count, cell volume), you must adequately dilute your

sample and repeat the measurement.

Measuring pore blockages

During the measurement, CASY® exactly monitors the rise times of the fluid column. This procedure ensures the

reliable detection of even film-like deposits (protein, lipids etc) in the measuring pore, which shrink rather than

actually block the pore, resulting in incorrect volume calculation. If the rise time exceeds a critical limit, the

following error message appears:




Measuring Timeout !

Capillary is possibly blocked. Further information will be provided by your manual.


Most blockages occurring during the measuring operation result from the temporary presence of very large

particles (e.g. aggregates, tissue remnants) in front of the measuring pore, which act like a “valve flap”. You can

repeat the measurement immediately after confirming the error message by pressing ENTER. Shaking the

CASY®cup slightly often helps to remove contamination from the outside of the measuring pore. If the blockage

remains, you can usually remove the contamination easily using the CLEAN function.

Caution:

During the CLEAN cycle, fluid is flushed under pressure from inside the measuring capillary through the measuring

pore into the sample cup. To prevent your sample becoming diluted or contaminated, replace it with a CASY®cup

containing freshly filtered CASY®ton before you run a CLEAN cycle.

If even repeated CLEAN cycles fail to remove a measuring pore blockage, you can unscrew the measuring capillary

(see 3.2) and try to flush the measuring pore through from the top using a syringe. If necessary, place the

measuring capillaries in CASY®clean and leave overnight.

Air bubbles

CASY® precisely monitors the incidence of “critical” air bubbles in the rising fluid column within the vertical tube.

Large air bubbles affect the accuracy of the sample volume measurement and may therefore result in the cell

concentration being measured incorrectly. If an air bubble exceeds a critical size during a measurement, the

following error message appears:




Large Air Bubble detected !

Regular use of CASYclean. is recommended.

Further information will be provided by your manual.


If you observe the vertical tube in the inspection window at the front of the measuring unit, you will be able to see

the air bubble. If the air bubble rises to the fluid meniscus and disappears, it will not affect subsequent

measurements. You can repeat the measurement immediately after confirming the error message by pressing

ENTER. If, however, the air bubble remains suspended in the vertical tube, you must remove it by replenishing the

fluid system using the CLEAN flushing function.

Deposits within the fluid system sometimes become noticeable through frequent air bubble error messages. If

necessary, run a CASY®clean cycle (see 5.1) to remedy the problem. Ensure that the system is flushed thoroughly

with distilled water to remove any remnants of CASY®clean!

Note:

If, during the measurement, only small air bubbles are evident within the vertical tube, no action is necessary. Small

air bubbles – which are higher in number with small measuring capillaries – are caused by the process of

electrolysis at the inner electrode. These are quite normal, no error message is triggered and the measurement is

not affected.

Empty Storage container

Should CASY® fail to completely replenish the fluid system during a CLEAN cycle, the following error message

appears:




Error-Code: PR-8

Liquid System could not be filled during Clean!

Please check, that there is enough CASYton in the storage container. Further information will be provided by your manual.

---------------------------------------- PRESS ENTER TO CONFIRM OR PRINT TO GET A HARDCOPY PRESS CLEAN TO FLUSH THE LIQUID SYSTEM.

Ensure that there is sufficient CASY®ton in the storage container (right hand container). Replenish the CASY®ton if

necessary and run another CLEAN cycle. If the error message re-appears even when the storage container is

sufficiently full, the pressure system may be mal-functioning or the internal tube system is blocked. In this case,

contact innovatis AG or your nearest dealer.

7.5. Setting the measuring range and the cursor positions

Accurate measuring results rely on the measuring range and cursor positions being correctly adjusted. It is

particularly important for evaluation of the data, that all samples to be compared are measured with identical

settings. It is generally advisable to specify the measuring range and the cursor positions for a particular cell type or

a certain application after the appropriate preliminary tests have been performed in a setup. For the required

preliminary tests, the cursors can be controlled manually using the control panel keys.

Depending on your application, completely different settings may be required. It is usually simple to derive the

correct setting logically from the displayed size distribution. If you have any questions regarding the settings for

your application, please contact the Application Support at innovatis AG. We will be pleased to help.

The settings for the measuring range and the cursors for monitoring the status of eucaryotic cells is described

below.



Measuring range

Select the measuring range so that even the largest anticipated cell aggregates can be recorded within the

displayed size distribution. For most eucaryotic cells, a measuring range of between 0-40 µm and 0-60 µm has

proven optimum. If you are not sure whether your cells will aggregate more intensely under different experimental

conditions, you should select a larger rather than a smaller range.

Evaluation and normalization cursors

To monitor the status of cell cultures, the right-hand cursors (evaluation and normalization cursors) must always be

positioned at the right-hand edge of the size distribution. This guarantees that all cell aggregates are included in

the calculations. To specify the position of the left-hand evaluation cursor and the left-hand normalization cursor,

you must first perform a measurement on a culture with high viability. Then kill an aliquot of your cells and

measure the size distribution of these dead cells. To finally set your cursors, it is recommended that you perform a

measurement on a mixture consisting of 70% viable cells and 30% dead cells. You should see a size distribution

similar to the one in the measuring example below.

• Set the left-hand normalization cursor at the lowest point on the size distribution between cell debris and

dead cells.

• Set the left-hand evaluation cursor at the lowest point on the size distribution between dead and viable cells.

This setting should ensure that all measuring results and calculations for this cell type are correct. When you have

created the correct settings for your various cell types, you should save them as setup and assign them a typical



name. This guarantees that the same cell type will always be measured with the same settings, i.e. that the

measurements are and remain comparable.

7.6. Manual cursor control using control panel keys

You can use the manual cursor control keys located on the left-hand side of the control panel to change the

position of the cursor whilst visually monitoring the size distribution. If you are in the numerical results display or

menu control, you must first move to the display.

Using the L-CURSOR and R-CURSOR keys activates the mode to manually set the cursor. This mode is identified

by the presence of a in the top right-hand corner of the screen, just before the cursor position to be adjusted

using the arrow keys. The evaluation cursor setting is always active after starting manual cursor control.

Alternate between L-Cursor and R-Cursor to activate the setting for the left and/or right-hand evaluation cursor.

You can switch to the normalization cursor setting by pressing NEXT. Now you can use the L-Cursor and R-

Cursor keys to activate the adjustment for the left and right-hand normalization cursor respectively. Pressing NEXT

again returns you to the evaluation cursor setting.

The and keys move the active cursor right and left respectively. The selected cursor moves across the

size distribution accordingly. At the same time, the numerical display of the cursor position is constantly updated in

the top right-hand corner of the size distribution. Pressing L-Cursor, R-Cursor and NEXT enables you to select a

different cursor to move at any time.

The manual cursor setting mode can be terminated by pressing either ENTER or CANCEL. If you press ENTER, the

new cursor positions are accepted as active settings and the calculation of the displayed measured values is

updated. If you press CANCEL, the previous cursor position settings are restored. All new changes are rejected.

Note:

While you remain in the manual cursor control mode, only the L-Cursor, R-Cursor, , , CANCEL and

ENTER keys are active. You must terminate manual cursor control before you can use other control panel

functions.



7.7. Outputting the results

The data can be output via either a parallel or a serial interface. The parallel interface is provided to connect a

PCL-capable printer (PCL = Printer Control Language). All Hewlett-Packard printers and compatible products

satisfy this requirement (see Appendix 8.3., Technical Data). The data can be transferred to a computer for further

editing via the serial interface (see Appendix 8.2, Data output via the serial interface).

To export size distributions and measuring results into MS-Excel™ (PC) through a fully automated process, you will

require CASY®excell, a program available from innovatis AG.

If you have set the Print Mode parameter to Auto, the data are automatically output after each measurement via

interfaces specified in the current parameters (see 6.4). If Print Mode is set to Manual, you must start to output

the measuring results by pressing PRINT.

The Graphic parameter applies only to the printed output. Depending on whether the Graphic parameter is set to

On or Off, only the numerical results or both the numerical results and the size distribution are output. The

numerical output has the following format:

CASY Model TT Date : 15.04.02 Serial Number : TT-2-AX-9999 Time : 10:15 Setup : Tumor-03 Comment : _________________________________________________________________________________ User/Siganture : _________________________________________________________________________________ Capillary : 150 µm Conc. Range : OK Volume/ml : 1.431e+07 fl X-Axis Scale : 40 µm Counts : 2911 Eval Cur. left : 12.80 µm Counts> 50µm/ml : 1.500e+02 Eval Cur. right : 40.00 µm Aggr. Correct : 4.249e+03 fl Mean Diameter : 21.10 µm Norm Cur. left : 7.60 µm Aggr. Fact. : 1.157 Mean Volume : 4.917e+03 fl Norm Cur. right : 40.00 µm Viable Cells/ml : 1.684e+05 Peak Diameter : 20.10 µm Sample Volume : 3 x 400 µl Total Cells/ml : 2.028e+05 Peak Volume : 4.250e+03 fl Dilution : 5.000e+01 % Viability : 83.04 % Interface : Parallel %Calculation : Viability Cell Debris/ml : 7.843e+04 Print Mode : Manual

The Comment and User/Signature rows are intended for hand-written comments on the measuring results and a

signature where appropriate.

The size distribution is inserted between the User/Signature and the Measuring Results rows if the Graphic

parameter is set to On.



Note:

This example shows the format of the numerical printout for settings %Calculation = %Via and Aggr.Correct =

Manual. Details on possible settings can be found in Section 6.4. Depending on your settings, some of the para-

meters visible in this example may not appear and/or alternative parameters are shown!

If you have selected Par or P + S as an interface, a printer must be connected to the parallel interface. If you have

omitted to switch on your printer or if it is not correctly connected, the following error message appears when you

try to output data.


Error-Code: CM-1

Printer does not respond! Be sure that your Printer is switched on and connected.

---------------------------------------- PRESS ENTER TO CONFIRM OR PRINT TO GET A HARDCOPY

Press ENTER to confirm the error message, switch the printer on and check that it is correctly connected. Having

eliminated the cause of the error, you can resume outputting the data by pressing PRINT.



8. Appendix

8.1. Error messages

CASY® permanently monitors all its system functions and reports error messages as soon as a fault is detected:


Error-Code: XX-##

Please shut off your CASY For at least 5 seconds. If error persists note the code and contact Schärfe System or your local distributor.

---------------------------------------- PRESS ENTER TO CONFIRM OR PRINT TO GET A HARDCOPY

Generally, an error code consisting of two letters followed by a hyphen and a one or two figure number is output

(e.g.: PR-7).

In special cases, a combination of up to 12 hexadecimal characters may be output, which are arranged in 3 blocks

separated by colons.

If you receive an error message, first try to switch off and then restart your CASY®. Errors can sometimes be

triggered by power supply faults. If no error message appears when you restart the system, no further problems

should occur. If the same error message appears during the self-test or measuring operation, you should note the

error code. You can also output the error message on the connected printer by pressing PRINT.

The following errors may be caused by false handling and can usually be easily cleared.

CM-1: Check that the printer is correctly connected and switched on.

PR-7 Check that the selected capillary setting corresponds to the measuring capillary in use.

PR-8 Check that the storage container is full of CASY®ton.



All other error messages that have an error code are caused by faults within the system and cannot be cleared. The

same applies to the errors listed above, if they cannot be cleared by the actions described. Please contact the

Service Department at innovatis AG or your nearest dealer to clarify further procedure.

8.2. Data output via the serial interface

You can transfer all the data relevant to a measurement via the serial interface to a connected computer. This data

includes all the measuring parameters specified in the active settings, the content of the 400 size channels and all

the values calculated from the current cursor settings.

To enable CASY® to output the measured data via the serial interface, the Interface parameter must be set to Ser

or P + S in the current setup. If the Print Mode is set to Auto, the data is automatically exported after every

measurement. If Print is set to Manual, the data is output by pressing PRINT on the control panel. The data is

exported in text format (ASCII) and the individual values are separated by CR/LF.

To be able to accept the data, your computer requires a free serial interface. The connection is established by a

straight serial transfer cable with a minimum of three signal lines (TxD, RxD and SignalGround).

CASY® uses the following Transfer parameters: Baudrate: 19200

Parity: None

Databits: 8

Stopbits : 1

To export size distributions and measuring results into MS-Excel™ (PC) through a fully automated process, you can

obtain the CASY®excell program from innovatis AG. The package also includes the serial connection cable

required for data transfer.



Format of serial data transfer

Row Name of Parameter Format Comments

1 Serial Number @@@@@@@@@@@@

2 Setup Name @@@@@@@@@@

3 Setup Number ## 00 - 19

4 Time ##:## 24 h format

5 Date dd.mm.yyyy oder mm/dd/yyyy depending on the selected format

6 Dilution Factor #.###e+##

7 Measuring Cycles ## 1 - 10

8 Sample Volume (µl) ###.## corrected based on volume calibration

9 Mode of Aggregation Correction # 0=Off, 1=Manual, 2=Auto

10 Manual Aggregation Correction (fl) #.###e+## single cell volume

11 Percent Mode # 0=Off, 1=Viab, 2=%Cnt, 3= %Vol

12 Debris Mode # 0=Off, 1=On

13 Capillary Diameter (µm) ###

14 X-Axis Scale (µm) ###

15 Left Normalization Cursor (µm) ###.##

16 Right Normalization Cursor (µm) ###.##

17 Left Evaluation Cursor (µm) ###.##

18 Right Evaluation Cursor (µm) ###.##

19 Y-Axis #### Auto or 10 - 7500

20 - 419 Counts in the 400 Size Channels #####

420 Counts ########## within evaluation cursor range

421 Concentration Range # 0=Ok, 1=Too High

422 Counts > Upper Scaling Limit/ml #.###e+##

423 Aggregation Factor ##.###

424 Counts/ml #.###e+## within evaluation cursor range*

425 Total Counts/ml #.###e+## within normalization cursor range*

426 Viable Cells/ml #.###e+## within evaluation cursor range*

427 Total Cells/ml #.###e+## within normalization cursor range*

428 Cell Debris/ml #.###e+## on left side of left normalization cursor*

429 Volume/ml in fl #.###e+## within evaluation cursor range

430 Total Volume/ml in fl #.###e+## within normalization cursor range *

431 %Counts ###.## based on normalization range=100%*

432 %Viability ###.## based on normalization range=100%*

433 %Volume ###.## based on normalization range=100%*

434 Mean Volumen in fl #.###e+## within evaluation cursor range

435 Peak Volumen in fl #.###e+## within evaluation cursor range

436 Mean Diameter in µm ###.## within evaluation cursor range

437 Peak Diameter in µm ###.## within evaluation cursor range

438 Check Sum XXXXXXXX 4 bytes hexadecimal

Depending on your setup settings, some data tagged with is output as not calculated.



8.3. Technical data on CASY® Model TT.

Function: Electronic Cell Counter + Analyser System for determining cell count and

cell size distributions, aggregation and viability. Dimensions: Height: 39 cm Width: 31 cm Depth: 39 cm Weight: 14.5 kg (including control panel) Ambient conditions: maximum operating temperature: 10°C to 40°C recommended operating temperature: 15°C to 32°C Power supply: 230 V~ / 50 Hz 115V~ / 60 Hz Power consumption: max. 112 VA Fuses (primary): 230V: 2 x 800 mAT

115V: 2 x 1.6 AT Fuses (secondary): SI1: T 160mA

SI2: T 1.6A SI3: T 400mA SI4: T 2.5 A SI5: T 400mA

Volume measuring tube Calibrated, opto-electronically controlled volume measuring tuben with and pressure system: no-maintenance, sensor controlled pressure system. Mercury-free. Measuring principle: Electrical resistance measurement in 1 MHz, low voltage field with pulse

area analysis by Schärfe System. Distinctive measuring channels: > 512.000.

Volume resolution: 1 in 512,000 Displayed size channels: 400 Measuring range: 0.7 µm to 120 µm Measurement dynamics: Ratio of smallest to largest particles that can be analysed simultaneously:

> 1 : 70,000 in terms of volume, . > 1 : 40 in terms of diameter

Typical analysis time: 10 sec Typical sample volume: 5 µl – 100 µl Measuring volume: 200 µl – 4 ml



Analysis/results: Analysis of the size distribution using two cell-specific, adjustable cursor

pairs: Cell count: absolute, relative -%-,

Cell viability: absolute, relative -%-, Cell aggregate: Aggregation factor, aggregation correction, Cell volume: Volume, mean volume, total volume (bio-mass) Interfaces: Serial, RS232 (DB9) Parallel (DB25)

Only EIC or UL-tested devices can be connected to the RS232 and printer interfaces.

Printer output: PCL (Hewlett- Packard compatible printer)

Printout of: Size distribution Numerical result Setups Error messages

Data export: ASCII, fully-automatic export into MS-Excel™ using CASY®excell Technical features: Permanent, highly stable calibration for biological media –

certified by Roche Innovatis AG, identical for all CASY® systems. Recording of measuring results, including date, time, setup name, device

serial number and all device settings. System self-test checking all measuring-specific parameters before and

after each measurement. Electronic monitoring of the measuring pore. Automatic flushing of the measuring pore. Automatic detection and display of operating and system errors.

Integrated service functions for fast error diagnosis. Fan-less system suitable for use in clean rooms.

Operation: Menu-driven operation and control of all functions through a movable

control panel with membrane keyboard and graphics display. All cell-specific device settings can be saved as setup. 20 setups per measuring capillary – each assigned a different name. Output of results and size distribution to display and printer. Export function for computers via the serial interface. Maintenance: If cared for on a regular basis, CASY® is virtually maintenance-free. If a

service becomes necessary or requested by the user, contact Roche Innovatis AG or your nearest dealer. Servicing may only be carried out by engineers who have been trained and approved by Roche Innovatis AG.

CASY® is a registered trademark of Roche Innovatis AG.

Subject to technical alteration, Reutlingen, April 2002.

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